Ion exchange process for separating proteins



' contiguous to their surfaces.

United States Patent Ofiice Patented Feb. 8, 1966 3,234,199 iflNEXQHANGE FROCESS FSR SEPARATING PROTEINS Alien F. Reid, 3145 Spur Trail,Dallas 34, Tex.

No Drawing. Griginal application Dec. 18, 1958, Ser. No. 781,200, nowPatent No. 3,073,747, dated Jan. 15, 1953. Divided and this applicationOct. 24, 1962, Ser. No. 232,388 The portion of the term of the patentsubsequent to Feb. 16, 1971, has been disclaimed and dedicated to thePublic 2 (Iiairns. (Cl. 260112) This invention relates to control ofsolubil-ities by ionexchange reactions and more particularly to varyingsalt concentrations in solutions by ion-exchange reactions to controlthe solubility of the solution. This case is a division of my co-pendingapplication Serial No. 781,200 filed December 18, 1958 (now Fatent3,073,747), which was a continuation-impart of my application Serial No.392,507 filed November 16, 1953 (now abandoned), which was acontinuation-impart of my application Serial No. 235,557 filed July 6,1951 (now Patent 2,669,559), which in turn was a continuationin art ofmy application Serial No. 20,583 filed April 12, 1948 (now abancloned).

It is an object of this invention to adjust the ion concentration in asolution of substances by ion-exchange reactions.

It is a further object of this invention to separate substances from asolution by varying the ion concentration in the solution.

It is more particularly the object of this invention to separateproteins whose soluhilities differ from one another in solutions ofvarious salt concentrations by adjusting the ion concentration of thesalts in the solutions.

The process of the present invention consists broadly in an exchange ofions between a solution and an ionexchange resin whereby selected ionsare removed from the solution. This removal changing the concentrationof the ions in the solution afiects the solubilities of the othersubstances in the solution and the difierenee in the solubilities isemployed to cause a separation of the substances.

lonexchange materials are solids which have the capacity of exchangingone ion for another in a solution For example, if RH is the acid form ofa cation-exchange resin in contact with a solution of sodium chloride:

And if ROl-l is the hydroxyl form of an anion-exchange resin in contactwith a solution of sodium chloride:

Many ion-exchange materials are available in granular form suitable forpacking in extended beds through which solutions may seep. To follow theapplication of such a column in adjusting ion concentration, consid rthe case of a long cation-exchange resin bed which has been equilibratedwith a solution 0.1 M to sodium. For simplicity, the effect ofhydrogen-ion concentration is neglected. In the top a solution of 0.2 Mto sodium is poured. The reaction (1) is displaced to the night withabsorption by the resin of sodium ion and release to the solution ofhydrogen ion in the attempt to restore equilibrium. The sodiumconcentration of the solution is reduced at the top of the column. Thissodium depleted solution progresses further down the bed, its sodiumconcentration approaching nearer and nearer to 0.1 M until it exits atthat concentration. More and more solution can be put through and haveits concentration adjusted to 0.1 M until such time as the bed capacityfor sodium under the operating conditions has become saturated farenough down so that the remaining layers of resin are not able to removeall of the excess sodium. A similar situation exists if theconcentration of sodium in the solution introduced had been less than0.1 M-sodium from the resin going into the solution to make up thediiference.

Similarly, an anion-exchange resin bed could adjust the concentration ofany anions in a solution progressing through it. Thus with a mixture ofthe two types of ion-exchangers, a solution can be adjusted toconcentrations of ions compatible with the capacity of the exchangers,if, prior to passage of that solution, the bed has been conditioned tobe in equilibrium with the ion concentration desired.

Many natural proteins are soluble in aqueous solutions or" commonchemical compounds and are ofttimes found in mixtures in the samesolutions from which one or several ofthem is desired to be removed. Asan example, we may consider the fractions of human blood plasma. Theseinclude proteins known as albumins which are soluble in distilled water,and various types of glob-ulins which are not soluble in distilled waterbut are soluble in various concentrations of solutions of sodiumchloride. When human blood serum is separated from the blood cells andfibrin, it contains these proteins in solutions which have a saltconcentration of approximately 0.17 mole per liter. The globtdinspresent can be precipitated and the albumin left in the solutions itmost of the salts are removed from this serum. Such fractionation isvaluable because the separated albumin fractions can be used medioinallyfor the treatment of shock, etc, and the separated globulin containsseveral firactions which are medioinally useful as immune sera and inthe treatment of hemophilic cases, etc. Because of the diiferen-tsolubilities of these components, it is evident that fractionation canbe attained by adjusting the concentration or" the nonproteinconstituents of the solutions. Up to the present time no practicalmethod has been advanced for adjusting those concentrations for thatpunpose which was satisfactory from the standpoints of expense and theprotection of the chemically and thermally sensitive products. In orderto adjust the concentration of these chemical constituents, I haveintroduced the solution at conservative temperatures to beds ofion-exchange resins which have been conditioned so that they will absorbions in the proper amounts and proportions to leave a concentration ofions in the solutions favorable for such fractionation.

The introduction of such an improved method for this fractionation isimportant since present methods, as well as being quite expensive,involve processes such as the use of very cold alcohol which decreasesthe potency and desirability of some of the products.

The following is a procedure which may be fol-lowed in one applicationof the principle of this invention:

A mixture of a cation-exchange resin and an anionexchange resin iswashed thoroughly with distilled water to reduce the salt concentration.This mixture of resins is placed in a series of columns and washedagain. The solution of proteins to be separated, such as defibrinartedblood plasma, is poured through these prepared columns. At the end ofthis treatment the ions which are taken by the resins in theion-exchange reactions are removed from the solution. The removal ofthese ions by changing the ion concentration of the solution changes thesolubility of one or more of the proteins in the solution with relationto the other proteins and consequently causes precipitation. Thedifie-rent proteins are precipitated at different points in the processas their solubilities are different in the various ion concentrations.

Applying this to human blood plasma, by removing salt from the plasma,the globulins are rendered insoluble in the solution and precipitated,leaving the albumin which is soluble in distilled water. Theprecipitated globulins may be centrifuged off from the treated solu tionand those which remain trapped in the columns dis solved out with asaline solution. The final solution which has passed through all thebeds contains no ap preciable globulins after centrifugation. Albuminwhich is left in the precipitated globulin fractions may be dissolvedout with distilled water without dissolving the globulins. The globulinfractions which have been washed out of the columns with the salinesolution may contain some albumin because of the hold-up of liquid inthe columns under normal operation.

The globulin fractions are separated from each other by a process ofpreferential leaching and precipitation. The mixture is first washedfree of albumin and then leached with solutions of sodium chloride at acontrolled pH. Under these conditions one globulin sub-fraction isdissolved preferentially from the others. The salt concentration and thepH of the leaching solution is then adjusted with an ion-exchange resinmixture and a selective precipitation of globulins is obtained. Thisglobulin precipitate is subjected to another selective leaching followedby a selective precipitation. In this way, using proper control of thepH, the sodium chloride concentration and the concentration of othersalts, it is possible to fractionate the globulins to any degree ofrefinement. In the described separation of globulin and albumin in bloodplasma the mixture of two types of resins used is intended to keep thepH of the blood plasma solution from becoming either too alkaline or tooacid. This is achieved by having the cation-exchange resin remove thesodium ion in this separation replacing it with hydrogen ion and theanion-exchange resin removing the chloride ion replacng it with ahydroxyl ion. The subsequent neutralization of the hydroxyl ions withthe excess hydrogen ions and vice versa insures that no highconcentration of acid is present in the solution. This is important as ahighly acid pH may cause precipitation of the albumin and possiblydenaturation of the proteins, and a basic pH would denature theproteins. The other blood serum salts are removed similarly. In thepassage of the solutions through the ion-exchange resins some of theanion-exchange resin may be dissolved and taken into the solution.Pyrogens which are contaminants formed by bacteria may be present in thereconstituted plasma and could be present in the distilled water whichis used in the operations. Any bacterial matter which might also bepresent could form other pyrogens unless the operations were carried outat an elevated temperature which would be objectionable as causingdenaturation of the proteins. Therefore, to remove these impurities andcontaminants from the solution it is passed through two final bedscontaining only the cation-exchange resin. These beds are conditionedfirst with distilled water and finally with pyrogen-free water and thusare able to take up the aforementioned contaminants. Finally, thesolution is sent through a bacterial filter to effectively remove anysmall particles of resin or bacteria which might have been picked up.This is removed by reprocessing. The albumin in the solution afterconoentration is safe for intravenous use.

The following example of plasma protein fractionation will serve as anillustration:

A mixture of equal parts of Amberlite IR 100, cation exchange resin, andAmberlite IRA 400, an anion exchange resin which is a strongly basicquaternary ammonium type anionexchange resin prepared through theamination of a chloromethylated copolymer of styrene and divinylbenzenewith trimethylamine, both furnished by the Rohm and Haas Company,Philadelphia, is Washed thoroughly with distilled water until the saltconcentration is less than four parts per million. The Amberlite IR 100is in the hydrogen form and the Amberlite IRA 400 is in the hydroxylform. Defibrinated blood plasma is mixed with this mixture. The saltsare removed by the resins to a concentration of less than .001 ionicstrength and the mixture of the resins maintains the pH between 7.4 and5.5. Under these conditions most of the globulins precipitate out of thesolution leaving essentially all of the albumins in solution.

A further application of the process of this invention is shown inremoving hemoglobin from albumin. In samples of albumin a certain amountof hemoglobin which is released by the fracture of red blood cells ispresent and soluble in salt-free solutions. It is highly desirable toremove the hemoglobin, a highly colored impurity, from the albumin. Theiron in hemoglobin may be bound to salicylate at values of pH between 7and 5.5, conditions which will not precipitate albumin. By adjusting theconcentrations of sodium salicylate and acid ions appropriately and thenadding a mixture of cation and anion exchange resins which reduce theionic strength of the solution while maintaining the pH between 7 and5.5, the proteins from the hemoglobin contaminant are rendered insolubleand may he removed by centrifugation.

Another application of this invention is the separation of some of theproteins in milk. Here a sample of concentrated milk whey containinglactoalbumin and lactoglobulin may be passed through a washed mixture of70% Zeocarb H, an organic cation-exchange resin of the coal derivativetype, furnished by the Permutit Company, and 30% Amberlite IR 4B, anamine type resinous anion exchanger containing approximately 14%nitrogen in the hydroxyl form, furnished by the Resinous ProductsChemical Company. The mixture maintains the pH between 5 and 7. Thesalt-free solution contains primarily la-ctoalbumin, globulins beingprecipitated out as the salt concentration goes down.

Another example is in the separation of the globulins from the albuminin bovine serum. This is important as bovine albumin is routinely usedin the medical sciences as a diagnostic aid. This separation may beaccomplished by using either a mixture of four parts of Amberlite IR100, a phenol-formaldehyde resin with a polyhydric phenol base andcationically active -SO H groups, and nine parts of Permutit Deacidite,a highly basic aliphatic amine type anion-exchange resin; or by using amixture of one part of Amberlite IR and two parts of Amberlite IRA 400,a strongly basic anionexchange resin with a high stability. When theconcentration of the salts has been brought down to an ionic strength of.015 and has been maintained at a pH between 8 and 5 by treating thesolution with a resin mixture, most of the globulins precipitate outleaving a solution which may be satisfactorily purified by a subsequentheat treatment.

Another application of the invention is the separation of proteinsproduced by the action of bacteria on culture media. These proteinproducts may be used, when modified somewhat chemically, as antigens toproduce immunity in the body because of their stimulation of theproduction of antibodies. An example of this is diphtheria toxoid. Inthe production of this toxoid, Corynebacterium diphtheriae is allowed togrow in a culture medium. The toxicity of the resulting protein isdestroyed by treatment with formaldehyde or some other modifying meanswhile still preserving the antigenicity of the material. This detoxifiedprotein must then be freed from other colloids present in order to makea preparation which is satisfactorily active and safe for injection. Itis found that after the gross impurities have been separated from thetoxoid, it may be further purified by subjecting its solution to amixture of two parts Amberlite IRA 400 to one part Amberlite IR 100,thus reducing the salt concentration to below an ionic strength of .001while keeping the pH value between 6.5 and 7.5. This treatment causesprecipitation of contaminating colloid material which may then bemechanically separated from the rest of the proteins. The resultingprotein may be even further separated by adjusting the pH with AmberliteIR 100 or a mixture of Amberlite IR 100 and a smaller proportion ofAmberlite IRA 400 to a value of 3.5 where an electrophoretically purecomponent is precipitated out. This may then be separated from the restof the solution by mechanical separation.

A similiar application of the invention exists in the purification oftetanus toxoid. After production in a similar manner by the action ofClostridium temm' on a nutrient medium and gross separation of theprotein, the toxoid containing fraction may be purified from otherprotein colloids by removal of the salts to an ionic strength of 0.001,keeping the pH value between 6.5 and 7.5 using a mixture of two parts ofAmberlite IRA 400 to one part Amberlite IR 100. The precipitatedimpurities may be removed'by mechanical separation. The remainingsolution may be further separated by reducing the pH with Amberlite IR100 or with a mixture of Amberlite IR 100 and a smaller proportion ofAmberdiscrete protein component will precipitate out. This discreteprotetin component will precipitate out. This may be removed bymechanical separation.

Two important by-products of the meat packing industry are the enzymespepsin and rennin. They occur in the gastric secretions of such animalsas cattle and hogs and are recovered from these sources. In the state inwhich they are recovered in the crude secretions they are not practicalfor commercial sale or usage. It is, therefore, necessary to purifythem. A further application of this invention may be utilized in thispurification. It is found that by using a mixture of about one partAmberlite IRA 400 to one part Amberlite IR 100 for the removal of saltsand acid to an ionic strength of .001 and a pH of 3, many of the proteinimpurities are precipitated out from a pepsin solution. These may beremoved by mechanical separation. On further treatment of the pepsinsolution with Amberlite IRA 400 to bring the pH to 3.52, veryconcentrated pepsin will precipitate out. The mixture of Amberlites IRA400 and IR 100 serves to keep the pH low enough so that the pepsin,which is unstable at a high pH, is not denatured.

As another modification of the procedure of the ap plication of thisinvention, the reduction of the salt con centration of the solution ofpepsin may be accomplished by first subjecting the solution to a smallamount of Amberlite IR 100, not allowing the pH to go below 1, thentransferring the solution to a small amount of Amberlite IRA 400, notallowing the pH to rise above 3, then transferring the solution toanother batch of IR 100, then to another batch of IRA 400 and so on,always keeping the pH between the limits of l and 3 until the ionicstrength has been reduced to 0.001. The solution is finally treated withAmberlite IRA 400 to bring the pH to 3.52. This treatment causesprecipitation of concentrated pepsin as in the example above.

In a similar application of the invention, if a crude rennin solution ata neutral pH is treated with a mixture of two parts Amberlite IRA 400and one part Amberlite IR 100, the salt concentration may be brought toan ionic strength of .001 keeping the pH between 6.0 and 7.5. Manycontaminating proteins are precipitated upon such treatment and may beremoved by mechanical separation. Upon reduction of the pH to 3.75 bytreatment with Amberlite IR 100 or a mixture of Amberlite IR 100 andAmberlite IRA 400, a concentrated rennin is precipitated.

As another modification of the procedure of the application of thisinvention, the reduction of the salt concentration of the solution ofrennin may be accomplished by first subjecting the solution to a smallamount of Amberlite IR 100, not allowing the pH to go below 5, thentransferring the solution to a small amount of Amberlite IRA 400, notallowing the pH to rise above 8, then transferring the solution toanother batch of IR 100, then to another batch of IRA 400 and so on,always keeping the pH between the limits of 5 to 8 until the ionicstrength has been reduced to .001. Finally the solution is brought to apH of 3.75 by treatment with Amberlite IR or a mixture of Amberlite IR100 and Amberlite IRA 400. This treatment causes precipitation ofcontaminating colloid material as in the example above.

In the above described preparations, theenzymes retained their originalactivity. In other methods of removal or separation, such as theprecipitation by saltingout with a high concentration of sodiumsulphate, these enzymes may be denatured.

Separation of the proteins of peanuts, urease and wheat fiour has beenefiective at pH values below 6. Many natural proteins are of vegetableorigin and are quite important in the economic life of today,particularly in the food industry. For example,,it is found that thetype of proteins present in ordinary wheat flour are very important inthe useof this flour for the manufacture of products such as bread,cake, etc. Attempts to modify the concentrations of the proteins inflour -so as to be able to market it for certain uses have been defeatedby the failure to separate out some fractions from other proteinconstituents by a practical means. An application of this invention tothis separation is to solubilize a portion of the wheat proteins in asalt solution and then to precipitate out certain proteins by removal ofthe salts using ion exchange resins. In a specific instance, somecomrnonwheat flour was mixed with a solution of salt with an ionic strength ofapproximately 0.15. The resulting solution of wheat proteins wasmechanically separated from the remaining solid material and subjectedto a mixture of one part Amberlite IRA 400 and two parts of Amberlite IR120, a strong cation exchange resin. As the salt concentration decreasedwith the pH also decreasing from 7 to 5, wheat proteins fractionallyprecipitated out. These could be mechanically separated from thesolution.

Other natural vegetable proteins of considerable food value are found inpeanuts. After the oil was removed from some crushed peanuts, some ofthe proteins were dissolved in a salt solution of an ionic strength ofapproximately .15. Part of these proteins were then fractionallyprecipitated by reducing the salt concentration to an ionic strength ofless than .07 using a mixture of one part of Amberlite IRA 400 to onepart of Amberlite IR with the pH being reduced from 7 to 4.5.

Certain vegetable proteins have enzymatic activity. One of the mostimportant of these. is urease which is made from jackbeans. After thisprotein has been separated grossly from the inactive material accordingto the most advanced means in common practice, the enzyme is stillcontaminated with much protein which serves not only to dilute itsactivity but also actually to inhibit some of the enzyme action. If someof this grossly purified urease is placed in a solution of salt with anionic strength of .15 and then subjected to a mixture of one part ofAmberlite IRA 400 to one part of Amberlite IR 1 20 and the saltconcentration brought to lower and lower ionic strength while the pH isbrought down to between 6.5 and 4.5, some relatively inactive proteinswill be first precipitated and later the urease will be precipitated ina much purer form.

In all of the instances cited, a modification of the process is toalternately treat the solution with the cation exchange resin notallowing the pH to go below the lower limit, and treat it with the anionexchange resin not allowing the pH to go above the upper limit,continuing the alternation until the ionic strength and pH have reachedthe desired values.

I claim:

1. A process of separating a protein from other proteins in an aqueoussolution containing a plurality of proteins as solutes and also having asalt content present as cations and anions in a concentration renderingsaid first-mentioned protein soluble in said solution and in the absenceof which cations and anions said protein is insoluble in said solution,another protein solute of said solution being soluble therein in theabsence of said cations and anions in said concentration, comprising thesteps of providing a mixture of cation-exchange and anion-exchangeresins conditioned to be in equilibrium With an ion concentration atwhich said solution-contained cations and anions are removed from saidsolution being treated so that said resins will sorb from solution saidcorresponding ions, passing said aqueous solution of said proteins andsalt solute in contact with said cation-exchange resin and saidanion-exchange resin adjusting the pH of the solution to be in the rangepH 1 to less than pH 6 by ion-exchange between said solution and saidresins, removing from said solution said solubilizing cations by ionexchange with said cation-exchange resin to reduce said solubilizingcations and removing from said solution said solubilizing anions by ionexchange with said anionexchange resin to reduce said solubilizinganions, and thereby precipitating said first-mentioned protein from saidsolution of said proteins, and separating said precipitate from saidsolution.

2. A process for the separation of certain proteins from other proteinsin a blood solution selected from the group consisting of blood plasmaand blood serum and aqueous solutions of natural blood proteins andcontaining an adequate concentration of the natural anion and cationsolubilizing ions, the said certain proteins being solubilized by saidions in the blood solution and the other said proteins being bothsoluble in the blood solution and in the absence of said concentrationof said solubilizing ions, comprising the steps of providing, a mixtureof an anion-exchange resin and a cation-exchange resin which are capableof removing said solubilizing ions from said blood solution, passingsaid solution in contact with said anion-exchange resin and saidcation-exchange resin, holding the pH of said blood solution betweenlimits effective to work the desired separation and in a range of pH 1to less than pH 6 by ion exchangewith said anion-exchange resin and saidcation-exchange resin, removing from a portion of said solution bothsaid anion solubilizing ions and said cation solubilizing ions from saidblood solution by ion exchange with said anionexchange resin and saidcation-exchange resin to provide a reduction of both said anion andcation solubilizing'ions in said portion thereby precipitating therefromsaid certain proteins in the blood solution and then separating theprecipitated proteins from said solution retaining the said otherproteins.

References Cited by the Examiner UNITED STATES PATENTS 2,240,116 4/1941Holmes 260 -11 8 2,461,505 2/ 1949 Daniel 26O-1l2 2,669,559 2/1954 Reid260112 3,073,747 1/1963 Reid 260112 WILLIAM H. SHORT, Primary Examiner.

1. A PROCESS OF SEPARATING A PROTEIN FROM OTHER PROTEINS IN AN AQUEOUSSOLUTION CONTAINING A PLURALITY OF PROTEINS AS SOLUTES AND ALSO HAVING ASALT CONTENT PRESENT AS CATIONS AND ANIONS IN A CONCENTRATION RENDERINGSAID FIRST-MENTIONED PROTEIN SOLUBLE IN SAID SOLUTION AND IN THE ABSENCEOF WHICH CATIONS AND ANIONS SAID PROTEIN IS INSOLUBLE IN SAID SOLUTION,ANOTHER PROTEIN SOLUTE OF SAID SOLUTION BEING SLUBLE THEREIN IN THEABSENCE OF SAID CATIONS AND ANIONS IN SAID CONCENTRATION, COMPRISING THESTEPS OF PROVIDING A MIXTURE OF CATION-EXCHANGE AND ANION-EXCHANGERESINS CONDITIONED TO BE IN EQUILIBRIUM WITH AN ION CONCENTRATION ATWHICH SAID SOLUTION-CONTAINED CATIONS AND ANIONS ARE REMOVED FROM SAIDSOLUTION BEING TREATED SO THAT SAID RESINS WILL SORB FROM SOLUTION SAIDCORRESPONDING IONS, PASSING SAID AQUEOUS SOLUTION OF SAID PROTEINS ANDSALT SOLUTE IN CONTACT WITH SAID CATION-EXCHANGE RESIN AND SAIDANION-EXCHANGE RESIN ADJUSTING THE PH OF THE SOLUTION TO BE IN THE RANGEPH1 TO LESS THAN PH6 BY ION-EXCHANGE BETWEEN SAID SOLUTION AND SAIDRESINS, REMOVING FROM SAID SOLUTION SAID SOLUBILIZING CATIONS BY IONEXCHANGE WITH SAID CATION-EXCHANGE RESIN TO REDUCE SAID SOLUBILIZINGCATIONS AND REMOVING FROM SAID SOLUTION SAID SOLUBILIZING ANIONS BY IONEXCHANGE WITH SAID ANIONEXCHANGE TO RESIN TO REDUCE SAID SOLUBILIZINGANIONS, AND THEREBY PRECIPITATING SAID FIRST-MENTIONED PROTEIN FROM SAIDSOLUTION OF SAID PROTEINS, AND SEPARATING SAID PRECIPITATE FROM SAIDSOLUTION.